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. 2017 Aug 14;36(50):6873–6883. doi: 10.1038/onc.2017.280

Figure 6.

Figure 6

The UBF-dependent nucleolar sequestration of SmgGDS-558 protects SmgGDS-558 from proteasome-mediated degradation in the nucleoplasm. (a) Lysates from HEK293T cells expressing SmgGDS-558-HA and SmgGDS-558-NESmut-HA were immunoblotted using HA antibody. Two exposures of the same immunoblot are shown; SmgGDS-558-NESmut-HA was detected only in the long exposure. Immunoblotting with GAPDH antibody was used as a loading control (n=3). (b) HEK293T cells were transfected with SmgGDS-558-HA or SmgGDS-558-NESmut-HA, and 56 h later the cells were incubated with the indicated concentrations of MG-132 or lactacystin. After 16 h, cell lysates were immunoblotted using HA and GAPDH antibodies (n=3). Long and short exposures of the immunoblots are shown. Mean normalized densitometry values are shown in Supplementary Figure S6. (c) HEK293T cells expressing SmgGDS-558-HA or SmgGDS-558-NESmut-HA were incubated with cycloheximide (CHX; 1 μg/ml) for the indicated times, and cell lysates were immunoblotted using HA and GAPDH antibodies (n=3). (d) Mean densitometry values obtained from three independent experiments shown in c were used to fit exponential regression curves and determine the half-life of SmgGDS-558-HA and SmgGDS-558-NESmut-HA. (e, f) HEK293T cells transfected with SmgGDS-558-NESmut-HA (e) or SmgGDS-558-HA (f) were co-transfected with non-targeting (NT) siRNA or UBF siRNAs, and 56 h later the cells were treated with or without MG-132 (5 μm, 16 h; n=3). Cell lysates were immunoblotted using HA, UBF and GAPDH antibodies. Mean normalized densitometry values are shown in Supplementary Figure S6.